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No WB bands for H2AX at all...!

WB H2AX bands

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10 replies to this topic

#1 kokoakash

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Posted 13 August 2012 - 10:35 PM

Hi,
I am a PhD scholar working with HL-60 cells and I am trying to induce DNA damage to them and detect gamma H2AX proteins (15kDa) by western blotting using a well-documented toxin, Arsenic.
However, instead of seeing a band at the expected 15kDA region, I've detected nothing after repeating the experiment several times. However, my actin band appeared whenever I detected for H2AX and actin simultaneously but surprisingly, there were no band at all for H2AX...! Only once i got some light and merged bands at 15kDa but at that time no actin band appeared..!Posted Image
I am very sure that the H2AX proteins are in the cells because I am using high molar concentration of arsenic that is sufficient to induce DNA damage.

where i am going wrong: Posted Image
Running the gel at wrong voltage (100V) and time (2.5hrs)?
Using wrong PVDF membrane size (0.2uM )?
Transferring proteins at wrong current (200) or for wrong time (1h and 15min)
Using wrong dilution of primary antibody (1:500)?
Using wrong dilution of secondary antibody (1:10000)?


Here is my protocol:
1. Lyse the cells. centrifuge at top speed for 30mins at 4deg Celsius to remove cell debris.
2. Quantitate amount of proteins and load 20ul of protein sample onto a 13% gel. Run gel at 100V for 2.5hrs.
3. Transfer proteins to a 0.2uM PVDF membrane at1h and 15min.
4. Block membrane in freshly prepared 5% milk (in TTBS) at room temp for 1 hr.
5. Incubate membrane with anti-phospho-histone H2AX, diluted 1:500, overnight at 4deg Celsius.
6. Wash with TTBS (3X). Incubate with Rabbit HRP conjugated IgG (diluted 1:10000) for 1hr at room temp.
7. Wash with TTBS (3X) and detect with Immobilon Western Chemiluminescent HRP Substrate.

Please help me out with this problem of mine..Posted Image
I shall be very thankful to you.

Edited by kokoakash, 13 August 2012 - 10:36 PM.


#2 ascacioc

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Posted 14 August 2012 - 01:36 AM

What kind of transfer are you using: semidry or wet? It might be the case that yur protein is too small and is blotted through the membrane while actin is large enough to remain on the membrane. Add a second membrane on top of your membrane to check whether your protein is transfered through the first one. Do the antibody detection for both of them. Do you activate your PVDF with methanol before preparing the transfer sandwich?

Andreea

#3 lsek

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Posted 14 August 2012 - 02:10 AM

Hi kokoakash,

Size of the histone protein is very small. You can try to transfer the protein at shorter time, e.g. 10 to 20 min which will be more than sufficient. Some people double layer the PVDF as precaution.

One very important note, histones are quite basic (positively charged). Although residual SDS should be able to neutralize this and render it negatively charged during SDS-PAGE, the amount of SDS is not sufficient/diluted during the transfer step and hence reverting the histones back to positively charged. If that is the case, you will sometimes see a faint histone band or not at all. To circumvent this problem, reverse the position of the PVDF during transfer, or you can wedge the SDS-PAGE gel with PDVF at both sides of the gel for this purpose (one for capturing the histone at the cathode while the other is to trap the actin protein at the anode).


Useful reference: http://www.millipore...proteintransfer

Good luck.

===><===


#4 kokoakash

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Posted 14 August 2012 - 05:35 PM

What kind of transfer are you using: semidry or wet? It might be the case that yur protein is too small and is blotted through the membrane while actin is large enough to remain on the membrane. Add a second membrane on top of your membrane to check whether your protein is transfered through the first one. Do the antibody detection for both of them. Do you activate your PVDF with methanol before preparing the transfer sandwich?

Andreea

Hi kokoakash,

Size of the histone protein is very small. You can try to transfer the protein at shorter time, e.g. 10 to 20 min which will be more than sufficient. Some people double layer the PVDF as precaution.

One very important note, histones are quite basic (positively charged). Although residual SDS should be able to neutralize this and render it negatively charged during SDS-PAGE, the amount of SDS is not sufficient/diluted during the transfer step and hence reverting the histones back to positively charged. If that is the case, you will sometimes see a faint histone band or not at all. To circumvent this problem, reverse the position of the PVDF during transfer, or you can wedge the SDS-PAGE gel with PDVF at both sides of the gel for this purpose (one for capturing the histone at the cathode while the other is to trap the actin protein at the anode).


Useful reference: http://www.millipore...proteintransfer

Good luck.

Thanks, well I use wet transfering. I am greatly thankful to you for your kind suggestion.
For cell lysis, I use to add some enzyme inhibitors in my lysis buffer (i.e. PMSF: Serine protease and Thiol protease inhibitor. Aprotinin:Trypsin and Chymotrypsin inhibitor. Leupeptin: Trypsin, Plasmin and Papain inhibitor). Now as i am working with a phospho-protein (H2AX), do I need to add phosphatase inhibitors as well? If yes which inhibitors i will need?

#5 almost a doctor

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Posted 15 August 2012 - 12:42 AM

Yes you should use phosphatase inhibitors. Sodium orthovanadate (Na3VO4) is one of the most commons (I think), not sure of concentration but I know that some commercially available buffers have it at 1mM.
Also, try to block with other agents (milk has phospho proteins in it and can affect your detection).

Do you have a positive control to confirm your antibody is working? Have you titrated your antibody?

#6 kokoakash

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Posted 20 August 2012 - 05:16 PM

OK..! today i am going to start my WB again... following your suggestions.... wish me luck Posted Image

#7 kokoakash

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Posted 04 September 2012 - 06:15 PM

well pals..! thanks for all ur suggestions as finaly i detected some BANDS..!!!Posted Image ......hurray

#8 ascacioc

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Posted 04 September 2012 - 06:28 PM

me likey :) and happy for you

#9 kokoakash

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Posted 05 September 2012 - 07:02 PM

Thanks Andreea..!

#10 Cyp19ab

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Posted 28 November 2012 - 01:26 PM

Koko,
Glad you overcame problem of no bands for H2AX antibody. Could you please post here what are the changes you made to make it work for you?

Thanks,

-Cyp19ab


well pals..! thanks for all ur suggestions as finaly i detected some BANDS..!!!Posted Image ......hurray


Edited by Cyp19ab, 28 November 2012 - 01:28 PM.


#11 memari

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Posted 29 November 2012 - 12:48 PM

First, some antibodies are not strong for their antigens. So you have to use more primary antibody.

Second, what kind of ECL have you used? There are there kinds, week, regular and sensitive.
Base on our experiences, week antibodies do not show any bands with even regular ECL, like

Luminata Crescendo Western HRP Substrate from Millipore
http://www.millipore.../tech1/00112798
http://www.millipore...LE/00112798.pdf


We use regular ECL to have less overexposed bands. But sometimes, you have to use Sensitive ECL like this one from BioRAD:
Immun-Star™ WesternC™ Chemiluminescence Kit #170-5070
http://www.bio-rad.c...uminescence-Kit

Third, how much antigen are there on the PVDF? the less antigen, the more amount of primary antibody should be used.
I have even used up to 1:250 from primary antibody to see a band for an antigen.

Forth, also there are three kinds of Autoradiography Film, week, regular and sensitive.
We use this "HyBlot CL Cat#E3012 from http://www.denvillescientific.com/ " because it has less background.

Edited by memari, 29 November 2012 - 12:56 PM.

-----
Babak Memari





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